DESCRIPTION: This application is a second amended version of a FIRST Award renewal application. Dr. Berman's long range goal is to identify and characterize genes required for proper telomere structure and function in Saccharomyces. Telomeres are required for chromosome stability and to protect the ends of chromosomes from degradation and loss. They have heterochromatin-like properties and repress closely linked telomere-associated genes, a property called telomere position effect (referred to as TPE). A number of gene functions involved in telomere structure and/or function have been identified largely by other investigators. These include, Rap1p (a well-studied telomere binding protein and transcription activator), Rif1p (a Rap1p interacting protein), Est1p (a component of telomerase), the Sir1-4 proteins, HHF2 (encoding histone H4) and Nat1p and Ard1p (components of an N- acetyltransferase). In many organisms, telomeres are known to associate with each other and localize to the periphery of the nucleus. This may also occur in yeast. Using mutant screens devised and undertaken during the previous grant period, Dr. Berman identified five novel genes required for the localization of Rap1p to the nuclear periphery. She named these genes RLF-1 through -5, for Rap1p Localization Factors. In her preliminary analysis of the mutant phenotypes, she finds that two of the mutations affect telomere track length, all affect TPE albeit to different extents, all produce a diffuse intranuclear distribution of Rap1p which is normally seen in a punctate perinuclear pattern, and none affect mating efficiency. Genetic analysis of the RFL genes has revealed several very interesting findings such as unlinked, non-complementation among some the RFL genes and suppression of some of the RLF genes by plasmid copies of genes like SIR3, SIR4, or ARD1 and NAT1. Taken together, these results suggest that the RLF gene products may interact as part of a telomere complex involving Rap1p and perhaps other gene products such as the Sir proteins. Consistent with this is Dr. Berman's work on the Rlf2p which has a predicted coiled-coil structure with sequence homology to mouse lamin B2, a structural component of the nucleus. Dr. Berman will use cytological methods, such as fluorescence in situ hybridization (FISH), to determine the localization of chromosomal telomere DNA within the Saccharomyces nucleus in wildtype cells throughout the mitotic and meiotic division cycle and compare this to the localization patterns seen in the RLF mutant strains. A coupled immunofluorescence/FISH protocol will be used to explore the co- localization of telomere DNA and Rap1p. The RLF1, -3, -4, and -5 genes will be cloned and sequenced and null alleles constructed and the mutant alteration in the original mutant alleles determined. A null allele of RLF2 has been constructed and found to have a somewhat different phenotype from the original mutant isolate, rlf2-1, which Dr. Berman found to be a nonsence mutation at codon 315 of the 606 ORF. Genetic analysis will be used to explore the possible interactions between the RLF gene products. For example, Dr. Berman will look for synthetic lethal phenotypes in double rlf null mutant strains and for multi-copy suppression of one rlf mutation by another RLF gene. Studies will be undertaken to characterize the interaction of the Rlf2p with itself and other nuclear proteins. For this, Dr. Berman will use genetic methods, such as the Two-Hybrid System, and physical methods like co-immunoprecipitation.